Additional file 5 of Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes

Additional file 5: Figure S1. The Can-Seq pipeline identifies exon- and splice site-located EMS-induced canonical nucleotide variants and no false positives. Following filtration, the frequency of the most abundant variant nucleotide is shown for every exonic and splice site nucleotide of AGO1 and THO6 in four and two Can-Seq libraries, respectively. AGO1 and THO6 were two of 32 candidate genes represented in Can-Seq libraries JC#4 and JC#5, and AGO1 was one of 17 candidate genes in Can-Seq libraries JC#6 and JC#3. Furthermore, the same 23 rtp mutants are represented in Can-Seq libraries JC#4, JC#5 and JC#6, and a different collection of 20 rtp mutants are represented in Can-Seq library JC#3. For further details of these Can-Seq libraries see Additional file 2: Table S2. EMS-induced canonical variant nucleotides (i.e. G→A; C→T) are shown if (a) at least 200 reads cover the position, (b) at least 30 reads contain the variant nucleotide, (c) at least five reads containing the variant nucleotide align in both the forward and reverse orientation, and (d) the frequency of the variant nucleotide is greater than the arbitrary threshold of 0.75%, which is indicated by the horizontal red lines. The non-zero variant nucleotides in AGO1 were subsequently detected as being homozygous (green) or heterozygous (blue) mutations in one of the rtp mutants that contributed to the Can-Seq library. Only the single nucleotide at position 1866 in the THO6 alignment was identified as a non-zero variant that passed the Can-Seq pipeline filters except it fell under the 0.75% threshold, and the rtp mutant carrying this variant nucleotide was not determined (orange). This variant detected in THO6 was the only non-zero variant nucleotide detected below the 0.75% threshold across all 47 candidate genes represented in our Can-Seq libraries, indicating that the Can-Seq pipeline identifies very few, if any, false positive variant nucleotides. Furthermore, this figure along with Additional file 2: Table S2, clearly demonstrates the reproducibility of the frequency of variant nucleotides between independent Can-Seq libraries representing the same rtp mutants and candidate genes. The Y axis shows the frequency of a variant nucleotide and the X axis shows the nucleotide position in AGO1 or THO6.